Besides proteins, nucleic acids have also been found to have catalytic activities in recent years. The catalytic active nucleic acids can be catalytic DNA/RNA, also known as DNAzymes/RNAzymes, deoxyribozymes/ribozymes, and DNA enzymes/RNA enzymes. Catalytic active nucleic acids can also contain modified nucleic acids. The catalytic activities of nucleic acid-based enzymes always depend on the presence of certain cofactors, for example, metal ions. Therefore, nucleic acid enzyme-based biosensors for these cofactors (e.g. biosensors for metal ions) can be designed based on the activities of the corresponding nucleic acid enzymes. On the other hand, nucleic acids may be selected to bind to a wide range of analytes with high affinity and specificities. These binding nucleic acids are known as aptamers.
Aptamers are nucleic acids (such as DNA or RNA) that recognize targets with high affinity and specificity (Ellington and Szostak 1990, Jayasena 1999). Aptazymes (also called allosteric DNA/RNAzymes or allosteric (deoxy) ribozymes) are DNA/RNAzymes regulated by an effector (the target molecule). They typically contain an aptamer domain that recognizes an effector and a catalytic domain (Hesselberth et al. 2000, Soukup and Breaker 2000, Tang and Breaker 1997). The effector can either decrease or increase the catalytic activity of the aptazyme through specific interactions between the aptamer domain and the catalytic domain. Therefore, the activity of the aptazyme can be used to monitor the presence and quantity of the effector. This strategy has been used to select and design aptazyme sensors for diagnostic and sensing purposes (Breaker 2002, Robertson and Ellington 1999, Seetharaman et al. 2001). DNAzymes and DNA aptazymes are the most attractive candidate for sensor development because DNA is much less expensive to synthesize and more stable than RNA. In addition, general strategies to design DNA aptazymes, by introducing aptamer motifs close to the catalytic core of DNAzymes, are available (Wang et al. 2002). High cleavage activity requires the presence of effector molecules that upon binding to the aptamer motif, can allosterically modulate the activity of the catalytic core part of the aptazyme.
In vitro selection methods can be used to obtain aptamers for a wide range of target molecules with exceptionally high affinity, having dissociation constants as high as in the picomolar range (Brody and Gold 2000, Jayasena 1999, Wilson and Szostak 1999). For example, aptamers have been developed to recognize metal ions such as Zn(II) (Ciesiolka et al. 1995) and Ni(II) (Hofmann et al. 1997); nucleotides such as adenosine triphosphate (ATP) (Huizenga and Szostak 1995); and guanine (Kiga et al. 1998); co-factors such as NAD (Kiga et al. 1998) and flavin (Lauhon and Szostak 1995); antibiotics such as viomycin (Wallis et al. 1997) and streptomycin (Wallace and Schroeder 1998); proteins such as HIV reverse transcriptase (Chaloin et al. 2002) and hepatitis C virus RNA-dependent RNA polymerase (Biroccio et al. 2002); toxins such as cholera whole toxin and staphylococcal enterotoxin B (Bruno and Kiel 2002); and bacterial spores such as the anthrax (Bruno and Kiel 1999). Compared to antibodies, DNA/RNA based aptamers are easier to obtain and less expensive to produce because they are obtained in vitro in short time periods (days vs. months) and with limited cost. In addition, DNA/RNA aptamers can be denatured and renatured many times without losing their biorecognition ability. These unique properties make aptamers an ideal platform for designing highly sensitive and selective biosensors (Hesselberth et al. 2000).
To assay DNA/RNAzyme or aptazyme activity, detectable labels are used. However, many of these suffer from significant drawbacks. Radioisotopes incur safety and disposal concerns, whereas fluorophores may undergo photo-bleaching and may also inhibit the biological activity trying to be assayed; organic dyes, such as that used in a cocaine-detecting aptamer system (Stojanovic and Landry, 2002) require high concentrations for visual detection and are matched to a specific aptamer only after costly trial and error.
Metallic particles overcome all of these difficulties. They can be used in small (nanomolar) amounts as detection agents with aptamers without any of the disadvantages of organic dyes. In sensors based on aptamers using metallic particles for color detection, the cleavage of a nucleic acid substrate by the aptazyme (upon binding of an effector) may be detected by color changes.
Typically, a DNA/RNAzyme- or aptazyme-based sensor has three parts:
(1) a nucleic acid enzyme (in the following description, nucleic acid enzymes will be referred to DNA/RNAzymes and aptazymes) and a co-factor, such as a metal ion that catalyzes substrate cleavage;
(2) a nucleic acid substrate for the nucleic acid enzyme, wherein interior portions of the substrate sequence is complementary to portions of the enzyme sequence; and
(3) particles attached to polynucleotides that are complementary to the 3′- and 5′-termini of the substrate.
To detect the target cofactor or effector, the complementary portions of the polynucleotides (the polynucleotides attached to the particles complementary to the 3′- and 5′-termini of the substrate strand, and the 5′- and 3′-termini of the nucleic acid enzyme complementary to interior substrate strand sequences) are annealed in the presence of a sample suspected of containing the targeted cofactor or effector. If the cofactor or effector is absent, the aptazyme is either inactive or shows substantially reduced activity, resulting in no or little substrate cleavage and thus aggregation of the particles. If the cofactor or effector is present, the enzyme becomes active and cleaves the substrate, preventing aggregate formation because the link between the particles is broken by enzymatic cleavage. Table 1 exemplifies such a system.
CleavageAggregationColor*YES (the target effector isDISPERSEDREDpresent)NONON-DISPERSEDBLUE*When gold particles are used for detection.
In the case of gold particles, the aggregated state displays a blue color, while the dispersed state (or the non-aggregate state) is red in color. The presence of the target analyte as a cofactor or effector can be detected based on the appearance of the color of the sensor system.
Since the degree of cleavage is reflected in the degree of color change, the target cofactor or effector concentration can be quantified. For example, simple spectrometry may be used for sensitive detection. Not only can color change be used for detection and quantifying, other results of the cleavage may be employed, such as precipitation. By replacing the aptamer domain with aptamers recognizing pre-selected effectors, calorimetric sensors for any desired effector can be easily made and used.
Based on previous work, a colorimetric biosensor for Pb(II) based on the DNAzyme-directed assembled of gold nanoparticles and a calorimetric biosensor for adenosine based on the aptazyme-directed assembled of gold nanoparticles have been designed (see, for example, U.S. application Ser. Nos. 09/605,558; 10/144,094; 10/144,679; and 10/384,497). Though highly sensitive and selective, this type of analytical sensor requires heating to above 50° C. for several minutes and cooling slowly to room temperature in 2 hours for detection.